Products of animal cell culture

Translational significance

In biomedical research, the use of animal and human cell cultures has become beneficial for diverse
applications. It provides indispensable tools for producing a number of products, including
biopharmaceuticals, mABs, and products for gene therapy. In addition, animal cell cultures provide
adequate test systems for studying biochemical pathways, intra- and intercellular responses,
pathological mechanisms, and virus production. Some of the applications of animal cell culture are
discussed below.

1. Antiviral vaccines

Animal cell culture technology has played an important role in the development of viral vaccine
production. The establishment of cell culture technology in the 1950s and the consequent
replacement of live animals for the development of antigens have led to considerable progress in
bioprocess technology. With the advent of DNA technology, molecular manipulation of viruses has
led to the development of a recombinant vaccine against hepatitis B virus (HBV) and several others
potential vaccines that are in the final phase of clinical trials.

Viral particles production by cell culture

Virus-like particles (VLPs) are highly effective as they mimic the overall structure of the virus;
however, these particles lack the infectious genetic material. Capsid proteins can aggregate to form
core-like particles in the absence of nucleic acids. These spontaneously assembled particles are
structurally similar to authenticate viruses and are able to stimulate B-cell-mediated immune
responses. In addition, VLPs stimulate a CD4-proliferative response and cytotoxic T-lymphocyte
response.

Viral particle production by cell culture differs from the production of molecules such as proteins,
enzymes, and toxins by bacteria or animal cells. The product formation may not be related to the
development or growth of a cell and may occur through secondary metabolic pathways, unlike virus
production, which does not result from secondary metabolic pathway. Virus production occurs after
the viral infection directs cell machinery to perform viral particle production.

Two stages are involved in viral production:

1. Cell culture system: This requires the development of an efficient system for conversion of the
culture medium substrate in the cell mass.

2. Virus production: This phase differs from the infection phase and has different nutritional and
metabolic requirements. A number of immortalized cell lines are used for the industrial production
of viral vaccines. Table 14.5 gives the cell lines used for vaccines.
Production of virus-like particles

Most of the existing classical vaccines for viral disease are either altered or chemically inactive live
viruses. However, incomplete inactivation of a virus or reversion of an attenuated strain can risk
infection in vaccinated individuals. Viruses with segmented genomes with a high degree of genetic
exchange can undergo re-assortment or recombination of genetic material with viruses of different
serotypes in the vaccinated host, which can result in the production of new variants of the virus.
Moreover, some live virus vaccines are teratogenic; for example, Smithburn neurotropic strain (SNS)
(Smithburn, 1949) and MP12-attenuated (Caplen et al., 1985) vaccine strains of the Rift Valley fever
virus. A new type of vaccine that does not present the typical side effects of an attenuated or
inactivated viral vaccine has been made possible with the development of rDNA technology.

VLPs resemble and mimic virus structure and are able to elicit a strong immune response without
causing harm. The major advantage of VLPs are,

 Their simplicity and nonpathogenic nature.

 They are replication-deficient as they lack any viral genetic information, thus eliminating the
need for inactivation of the virus. This is important as inactivation treatments lead to
epitope modifications.
 As the structural morphology of VLPs is similar to the virus, the conformational epitopes
presented to the immune system are the same as for the native virus particles.
 The immune response/antibody reactivity in the case of VLPs is significantly improved as
VLPs present conformation epitopes more similar to the native virus.
 VLPs also induce a strong B-cell response.
 For broader and more efficient protection, it is possible to adapt one or more antigens to
the multimeric protein structure.
 Another advantage offered by VLPs is that they significantly reduce vaccine costs as these
can elicit a protective response at lower doses of antigen.
Vaccines based on virus-like particles

The FDA has approved VLP-based vaccines for HBV and HPV. The HBV vaccine was approved in 1986
and the HPV one in 2006. To generate immunogenic VLPs, the S gene is cloned and expressed in a
eukaryotic expression host such as yeast or mammalian cells (e.g., CHO cell line). The mammalian
cell culture allows easy recovery because the cells are able to secrete the antigen HBsAg. The two
companies producing CHO-based vaccines are the French-based Pasteur-Merieux Aventis (Gene
Hevac B) and the Israeli-based SciGen (Sci-B-Vac). The Gene Hevac B vaccine contains the HBsAg S
protein and M protein, whereas Sci-B-Vac contains the M and L proteins.

Baculoviral vectors for vaccine production

Baculovirus is extensively utilized as an excellent tool for production of recombinant protein in insect
cells. Baculovirus infects insects in nature and is non-pathogenic to humans. In addition to insect
cells, baculovirus is capable of transducing a broad range of animal cells. Due to its biosafety, large
cloning capacity, low cytotoxicity, and non-replication nature in the transduced cells as well as the
ease of manipulation and production, baculovirus has been utilized as RNA interference mediators,
gene delivery vectors, and vaccine vectors for a wide variety of applications.

Human papilloma virus vaccine

Viruses of the Papillomaviridae family are known to induce lesions and warts and also cause cervical
cancer. Fifteen strains of Papillomaviridae are known to cause cervical cancer. HPV-16 is considered
a high-risk HPV type as the risk of cancer may be higher than for other high-risk HPV types. The two
virally encoded proteins of HPV are L1 and L2. L1 is the main capsid protein that forms the outer
shell of the virus. L2 is found in the interior of the viral particle and is less abundant. The
recombinant L1 VLP is able to induce neutralizing antibodies in animals. Gardasil (the first HPV
vaccine) was approved by the FDA in 2006. This vaccine is manufactured by Merck and Co., Inc.
Ceravarix, another HPV vaccine (manufactured by Glaxo Smithkline), was approved by the FDA in
2009. It uses the Trichoplusia ni (Hi-5) insect cell line infected with L1 recombinant baculovirus.

2. Recombinant therapeutic proteins

Proteins play a major role in carrying out biochemical reactions, transporting small molecules within
a cell or from one organ to another, formation of receptors and channels in membranes, and
providing frameworks for scaffolding. The number of functionally distinct proteins in humans far
exceeds the number of genes as a result of post-translational modifications. These modifications
include glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and
lipidation. The changes in protein structure as a result of mutation or other abnormalities often lead
to a disease condition. Protein therapeutics offer tremendous opportunities for alleviating disease.
The first therapeutic from recombinant mammalian cells was human tissue plasminogen, which
obtained market approval in 1986. At present, 60%–70% of all the recombinant therapeutic proteins
are produced in mammalian cells.

Main therapeutic proteins

The main therapeutic proteins can be divided into seven groups:

1. Cytokines

2. Hematopoietic growth factors

3. Growth factors

4. Hormones

5. Blood products

6. Enzymes

7. Antibodies

Most of the proteins have complex structures and undergo chemical modification to insure full
biological activity. Protein post-translation modifications (PTM) can happen in several ways. The
most widely recognized form of PTM is glycosylation, which involves extensive sequence processing
and trimming in the Golgi apparatus and endoplasmic reticulum. Eukaryotic cells are capable of
carrying out this type of modification and are thus preferred in biopharmaceutical processes.
Hamster, baby hamster kidney (BHK), and CHO cells are often the host cells of choice as
glycosylation patterns generated from these cells are more similar to human patterns. Table 14.7
lists various therapeutic proteins produced in animal cell lines.

Cytokines : Cytokines are proteins of the immune system that play a central role in immune
response. Cytokines are produced as a result of immune stimulus by various white blood cells.
Interferons (IFNs) were the first family of cytokines to be discovered and used as
biopharmaceuticals.

Interferons

Interferon’s are proteins produced by a cell infected by a virus, and provide protection to other
healthy cells from viruses. Interferon was discovered in 1957 when Issacs and Lindenmann observed
that virus- free fluid obtained from cultured cells infected with virus protected other cells from virus
infection. They called the substance present in these fluids, which interfered with virus infection,
interferon.
There are three major types of interferons:

(i) Interferon-α (INF-a; produced by leucocytes or white blood cells).

(ii) Interferon-β (INF-p; produced by fibroblasts).

(iii) interferon-ϒ (INF- ϒ; produced by stimulated T- lymphocyte cells, hence also called immune
interferon).

The mechanism of protection by interferons appears to be as follows. When interferon reacts with
the interferon receptors of a cell, the cell enters in a state called interferon-induced antiviral state. In
this state, a rapid degradation of mRNA occurs if the cell is infected by any virus.

Interferon induces in cells the production of 2, 5-adenosine polymerase. When such a cell is infected
by a virus, the 2, 5-A polymerase is activated to produce 2, 5 adenosine (2, 5-A), which in turn
activates pre-existing but inactive molecules of ribonuclease-L. Activated ribonuclease-L degrades all
mRNA (of host as well as virus origin) present in the cell bringing the protein synthesis to a halt in
such cells.

This interferes with multiplication of the virus so that virus infection is either stopped or sufficiently
slowed down to allow the production of adequate antibodies against the invading virus. The
protection due to interferons is nonspecific in that interferon induced by any one virus will provide
protection against all viruses.

It is possible that interferons modify ribosomes so that they no longer translate viral mRNAs,
although they are fully capable of translating mRNAs of the host origin. Interferons enhance the
cytotoxic activity of natural killer cells (NK cells), which are a type of lymphocytes identifiable as
large granular lymphocytes (LGL). NK cells are cytotoxic to some types of tumour cells. Interferons
are known to inhibit growth of some types of tumours; most of these tumors are also responsive to
chemotherapy.

Interferons, therefore, have been employed in treatment of the responsive tumors despite their
prohibitive cost (initially, $50 million/ g of commercial product). Interferons are produced from
human leucocytes isolated from donor blood and cultured in vitro, and from mouse fibroblast
cultures. The production scheme, in simple terms, is as follows. Large scale cell cultures are infected
with Sendai virus, and incubated for 24 h after which the supernatant (clear fluid) is collected,
centrifuged, and used for interferon isolation.

The amount of interferon recovered is relatively small (1 g interferon of low purity from leucocytes
separated from blood of about 90,000 donors), and the normal leucocytes are difficult to culture
preventing scaling up from relatively small inocula. These contributed to the enormous price of the
product.

In view of the value of and demand for interferons, intensive efforts were made to produce it in
genetically engineered organisms, e.g., E. coli, yeast mammalian cell cultures and even in plants
These efforts have drastically reduced the cost (to less than 10% of the initial price) and improved
the purity (by several orders of magnitude) of the product.
Applications of interferons

IFNα is used for treatment of hepatitis, and more recently has been approved for leukemia and
other types of cancers. IFNβ is used for treatment of multiple sclerosis and is marketed under the
names Avonex, Belaseron, and Rebif. IFNγ is used for the treatment of chronic granulomatous
disease. Interleukin is another kind of cytokine that helps regulate cell growth, differentiation, and
motility and is used as a biopharmaceutical. The recombinant form of IL-2 is used for the treatment
of renal cell carcinoma.

Growth factors

Growth factors are proteins that bind to receptors on the surface of cells to activate the cells for
proliferation and or differentiation. The different types of growth factors are TGF, insulin-like growth
factor, and EGF. The primary sources of PDGF are platelets, endothelial cells, and the placenta. Two
isoforms of this protein are present in the human body and both of these have one glycosylation site
and three disulfide bonds. Examples of growth factors used as biopharmaceuticals are the following:

1. Osigraft/Eptotermin alfa (bone morphogenetic protein) is used for the treatment of tibia
fractures, is grown commercially in CHO cells, and was first approved in 2001 in Europe.

2. InductOS/Dibotermin (bone morphogenetic protein) is used for tibia fractures and in spinal
surgery; it is also commercially grown in CHO cells. This product was first approved in Europe in
2002.

Hormones

Insulin, glucagon, gonadotropins, and growth hormones are the most well-known therapeutic
hormones. The first biopharmaceuticals that obtained approval by regulatory agencies were insulin
and recombinant human growth hormones. These were produced in microbial cells. The commercial
recombinant forms of the gonadotropin family of hormones are Gonal-F, Luveris, Puregon, and
Ovitrelle. All these are produced using CHO cells and are used for treating female infertility.

Therapeutic enzymes

A number of recombinant therapeutic enzymes are expressed in mammalian cells. Tissue

plasminogen activator (tPA) is a thrombolytic agent involved in dissolving blood clots. Recombinant
tPA is commercially is known as Alteplase and Tenectplase, which are used for the treatment of
acute myocardial infraction.

Fabry disease, a genetic metabolic disorder, is characterized by a lack of enzyme α-galactosidase A.

Fabrazyme (approved in 2001) is a recombinant α-galactosidase A and is produced by genetically
modified CHO cells.

Blood coagulation factors Hemophilia A is caused by the lack of blood-clotting factor VIII, hemophilia
B is caused by deficiency of factor IX, and hemophilia C by lack of factor XI. Factor VIII and IX are
proteins. The first recombinant factor VII products were Recombinate and Kogenate, which were
expressed in CHO and BKH cells, respectively. Recombinant factor FIX is commercially sold as
BeneFIX and is produced in recombinant CHO cells.
Antibodies

Therapeutic antibodies are used in the treatment of cancer, cardiovascular disease, infections, and
autoimmune diseases. In 2004, the antibody Avasin (Bevacizeimab) was approved for the treatment
of metastatic colorectal cancer. This antibody acts as an inhibitor of vascular endothelial growth
factor. Zenapax, another commercially available antibody, is used during prophylaxis for preventing
the rejection of transplanted organs. This is commercially grown in the NSO cell line and was
approved for human use in 1997.

Gene therapy

Importance of cell culture in gene therapy

Gene therapy involves the insertion, removal, or alteration of a therapeutic or working gene copy to
cure a disease or defect or to slow the progression of a disease, thereby improving the quality of life.
The human genome map was the first major step toward a new way of addressing human health and
illness. Gene therapy holds great promise, however, the task of transferring genetic material into the
cell remains an enormous technical challenge and requires ex vivo cell cultivation and adaptation
from the lab to a clinically relevant state. The development of animal cell culture technology is
imperative for advances in gene therapy.

Monogenic diseases caused by single gene defects (such as cystic fibrosis, hemophilia, muscular
dystrophy, and sickle cell anemia) are the primary targets of human gene therapy.

The first step in gene therapy is to identify the faulty gene. This is followed by gene isolation and
generation of a construct for correct expression. Integration of the gene followed by delivery of the
genetic material in vivo or ex vivo is crucial to the success of gene therapy. In in vivo therapy, the
genetic material is introduced directly into the individual at a specific site, and in ex vivo treatment,
the target cells are treated outside the patient’s body. These cells are then expanded and
transferred back to the individual at a specific site. The ex vivo technique involves gene therapy in
the cultured cells, which are expanded and subsequently transferred to the targeted tissue.

Clinical correlation

A number of clinical studies and trials for gene therapy have already been approved and are being
conducted worldwide. From 1989 up to the present, about 500 clinical studies have been reported;
70% of these studies are intended for cancer treatment.

The first product designed for gene therapy was Gendicine, a medication produced by Shenzhen
Sibiono Genetech, China. Gendicine is used for head and neck carcinoma treatment. The tumor 4
suppressing gene p53 in recombinant adenovirus expresses protein p53, which leads to tumor
control and elimination. SBN-cel is a cell line that was subcloned from the human embryonic kidney
(HEK) cell line 293 and has been used for the production of Gendicine.
Biopesticides

In recent years, biopesticides have gained importance due to increased concerns about
agrochemicals and their residues in the environment and food. Biopesticides provide an effective
means for the control of insects and plant disease, and they are environmentally safe. The biological
control of insect pests by another living organism (in order to suppress the use of pesticides) is an
age-old practice. Presently, a number of biological controls are being used as biopesticides. With the
high cost of chemical-based pesticides and the development of resistance to multiple chemical
pesticides, baculoviruses are one of the most promising biocontrols for insect pests and have been
increasingly used effectively against caterpillars worldwide. However, the major impediment in the
development of baculoviruses as biopesticides is the high cost and small volumes of in vitro
methods. Development of an in vitro production process for large quantities of baculoviruses at
comparable costs to chemical pesticides will help provide insect control that is safe, efficacious, cost-
effective, and environmentally safe.

Baculovirus production in animal cell culture

A number of factors are important for a successful commercial production of bioinsecticides:

1. Large-scale production of viruses at competitive costs.

2. Economic production of viruses (i.e., low cost for the media and running the culture).

3. Effective cell line with high virus per cell productivity.

4. With passage of the virus into cells, there is a loss of virulence and an increased risk of mutant
formation; this should be avoided.

5. The quality of the polyhedral produced in the cell culture should be comparable to those obtained
from caterpillars.

The insect baculovirus cell system offers a number of advantages. It produces recombinant proteins
that are functional and are immunologically active, as it is able to make post-translational
modifications. The recombinant system uses a powerful promoter polyhedron.

Monoclonal antibodies

The majority of antibodies available on the market today are produced in animal cell cultures.
Animal cells are preferred because they are capable of glycosylation and structural conformation,
which is essential for a drug to be productive. Hybridoma technology has been the most widely used
method for small- and large-scale production of mABs. However, these antibodies have limited
therapeutic applications since they produce an adverse immune response on repeated use.

A number of cell lines are now being used for the production of recombinant antibodies. The CHO
lines are the most commonly used. Other cell lines used are marine myelomas NSO, Sp 2/0, HEK-93,
and BHK.